Fibronectin (FN) is a plasma, cell surface, and extracellular matrix molecule that plays an important role in mediating platelet deposition onto subendothelium exposed by vascular injury. To date, two distinct receptors for FN have been defined on the platelet surface, glycoprotein IIb-IIIa (alphaIIb-beta3 integrin) and glycoprotein Ic-IIa (alpha5beta1 integrin). Both receptors recognize the arg-gly-asp sequence and adjacent flanking sequences within the cell binding domain of FN in a divalent cation-dependent manner. Although these two receptors appear to adequately account for the fluid phase binding of FN to platelets, recent studies in several laboratories, including our own, indicate that the two receptors do not adequately account for all of the physiologically relevant adhesion of platelets to solid phase FN. Another mechanism must exist. We have recently documented a novel, divalent cation-independent mechanism of platelet adhesion to the gelatin binding domain of FN, a domain not previously implicated in the cell adhesive activity of FN. The experiments outlined in this proposal are directed towards defining the molecular basis and physiologic significance of this new adhesive mechanism. We will define the minimum active recognition sequence within the gelatin binding domain by first identifying an active cyanogen bromide fragment and then using synthetic peptides to refine the recognition sequence. The function of the recognition sequence will be confirmed by demonstration of the adhesive activity of synthetic solid phase substrates containing the sequence, and by the ability of antibodies directed against the sequence (or adjacent flanking sequences) to inhibit the divalent cation- independent component of platelet adhesion to FN and to inhibit platelet adhesion to the gelatin binding domain. Photoaffinity labeling and affinity chromatography procedures will be employed to identify and purify the platelet surface receptor mediating adhesion to the gelatin binding domain. Receptor function will be established by examination of the adhesive activity of liposomes bearing the purified platelet receptor and by the development and use of inhibitory anti-receptor antibodies to inhibit platelet adhesion. The physiologic significance of this novel adhesive mechanism will be established in a series of experiments carried out in the rectangular perfusion chamber under conditions of flow using substrates composed of the purified 40 kDa domain, intact FN, collagen, and endothelial cell extracellular matrix and the inhibitory reagents directed against the minimal recognition sequence and the receptor. Lastly, we will ascertain whether the receptor (or immuno-chemically related protein) is expressed on the surface of other cell types and if other cell types expressing the receptor use it to adhere to the gelatin binding domain of FN. The strategy to be employed is patterned after that which served us well in elucidation of the role of the alpha2beta1 integrin as a cell surface receptor for collagen on platelets and other cells.